Salt of GL-V8 as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of medicine, and discloses a GL-V8 salt and a preparation method and application thereof, the GL-V8 salt is a salt formed by 5-hydroxy-7-(4-(bis (2-ethoxyl) amino)-1-yl) butoxy-8-methoxyflavone and pharmaceutically acceptable acid, and the stoichiometric ratio of the salt to the pharmaceutically acceptable acid is 1: 1; the salt of GL-V8 or the pharmaceutical composition thereof can be used for preparing drugs for preventing or treating hepatic fibrosis, sepsis, colitis or cancers. The compound has high in-vivo anti-tumor activity and is expected to become a novel medicine for treating leukemia.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to a specific salt form of GL-V8 and a specific salt form of the pharmaceutically active compound 5-hydroxy-7-(4-(bis(2-hydroxyethyl)amino)-1-yl)butoxy-8-methoxyflavone, including its amorphous and crystalline forms (including solvate and hydrate forms), and a preparation method and use thereof. Background Art

[0002] GL-V8 is a derivative of the flavonoid compound wogonin, with a molecular formula of C 24 H 29 NO7, chemical name is 5-hydroxy-7-(4-(bis(2-hydroxyethyl)amino)-1-yl)butoxy-8-methoxyflavone, its structure is shown in (Ⅰ):

[0003]

[0004] GL-V8 is a derivative of the flavonoid compound baicalin, which is the active ingredient in the traditional Chinese herbal medicine Scutellaria baicalensis. GL-V8 is one of the derivatives of baicalin and has the structural characteristics of a lysosomal-affinity molecule.

[0005] Studies have shown that GL-V8 has a wide range of pharmacological activities and has significant inhibitory effects on various tumor cells such as T lymphocyte leukemia, colorectal cancer, and pancreatic cancer in vivo and in vitro. Its initial mechanism of action is to mediate lysosome-dependent cell death through lysosomal damage, thereby selectively killing malignant tumor cells, and has no obvious toxic side effects on normal tissues and cells, which is not available in general chemotherapy drugs. GL-V8 is one of the derivatives of baicalin and has the structural characteristics of lysosomal molecules. LC-MS detection shows that GL-V8 can accumulate in high concentrations in lysosomes in a short period of time. The experimental results show that GL-V8 causes lysosomal damage to tumor cells in the early stage, increases the accumulation level of sphingomyelin in the lipid components of the lysosomal membrane, and reduces the activity of acid sphingomyelinase ASM, effectively inducing lysosome-dependent cell death in tumor cells and exerting an anti-tumor effect. We screened the target proteins of CPU-008 in lysosomes through a DARTS-based proteomics approach, preliminarily established HSP70 as the binding target of GL-V8, and used the CETSA method to verify the binding ability of the compound to the target molecule, suggesting that GL-V8 interferes with the stability of lysosomes by targeting and inhibiting HSP70.

[0006] The prior art discloses a variety of pharmacological effects of GL-V8 and its applications in a variety of pathological models, including anti-glioma (for example, Chinese invention patent publication number CN116999430A discloses a QL-H008 preparation for treating glioma, its preparation method and application patent), anti-melanoma (for example, Chinese invention patent publication number CN117243941A discloses the application of flavonoid derivative CPU-008 in the preparation of melanoma treatment drugs), etc.

[0007] In general, salts, solvates, hydrates, polymorphs, crystalline and amorphous forms of a given substance usually differ in crystal behavior and / or crystalline solid state properties, and therefore they may have different physical and pharmaceutical properties, such as shape, density, hardness, deformability, stability, purity, hygroscopicity, flowability, compactness, solubility and / or dissolution properties, etc., which may, for example, affect: their manufacturability, processability, pharmacokinetic properties (such as bioavailability), drug stability (shelf life), dosability and / or formulability, etc., such as their suitability as solid, semisolid or liquid pharmaceutical dosage forms (e.g., such as tablets, capsules, suspensions, solutions, suppositories or other pharmaceutical dosage forms).

[0008] Nearly half of drug molecules exist in the form of salts. Drugs can improve some undesirable physical, chemical or biopharmaceutical properties of drugs, such as changing the solubility or dissolution of drugs, reducing hygroscopicity, improving stability, changing melting points, facilitating preparation and purification, and improving permeability. It is necessary to select suitable salt forms for drug development. At the same time, a salt form may exist in different crystal forms. Different crystal forms have different melting points, solubility, dissolution properties, chemical stability, etc. These physical and chemical properties may directly affect the effectiveness of the drug. Therefore, salt form screening and crystal form screening are one of the important links in drug development. For a specific compound, the advantages and disadvantages of the physical and chemical properties of its free state, various salt forms and corresponding crystal forms are unknown. Based on further consideration of its drugability, finding suitable salt forms and providing a variety of intermediates and / or raw materials for subsequent drug development are of great significance to drug development.

[0009] However, there are no reports on GL-V8 salt forms, so it is necessary to conduct a comprehensive and systematic screening of multiple salt forms of GL-V8 to select the most suitable salt form for drug development. In addition, during the development of the production process of GL-V8, its free base has a strong electrostatic effect, which is not convenient for the expansion of preparation production. Summary of the invention

[0010] The object of the present invention is to solve the deficiencies in the prior art and provide an acid addition salt (especially hydrochloride) of 5-hydroxy-7-(4-(bis(2-hydroxyethyl)amino)-1-yl)butoxy-8-methoxyflavone having good pharmacodynamic activity and a preparation method thereof.

[0011] In order to achieve the above object, the present invention is achieved through the following technical solutions:

[0012] In a first aspect, the present invention provides a salt of GL-V8, wherein the salt of GL-V8 is a salt formed by 5-hydroxy-7-(4-(bis(2-hydroxyethyl)amino)-1-yl)butoxy-8-methoxyflavone and a pharmaceutically acceptable acid in a stoichiometric ratio of 1:1.

[0013] Inorganic acids which form pharmaceutically acceptable acid addition salts include, by way of example and not limitation, hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid, sulfuric acid, and the like.

[0014] Organic acids that form pharmaceutically acceptable acid addition salts include, by way of example and not limitation, acetic acid, 2,2-dichloroacetic acid, adipic acid, ascorbic acid (in its D- or L- form, especially in its L- form), aspartic acid (in its D- or L- form, especially in its L- form), benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, benzoic acid, 4-acetamido-benzoic acid, camphoric acid (in its (+)- or (-)- form, especially in its (+) form), camphor-10-sulfonic acid (in its (+)- or (-)- form, especially in its (+) form), decanoic acid (caprylic acid), caproic acid (caprylic acid), caprylic acid (caprylic acid), carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecyl sulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxy-ethanesulfonic acid, formic acid, galactaric acid , gentisic acid, glucoheptonic acid (in which D- or L- form, especially its D- form), gluconic acid (in which D- or L- form, especially its D- form), glucuronic acid (in which D- or L- form, especially its D- form), glutamic acid, glutaric acid, 2-oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid (in which D- or L- form), lactobionic acid, lauric acid, maleic acid, malic acid (in which D- or L- form), naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, palmitic acid, pamoic acid (pamoic acid), propionic acid, pyroglutamic acid (in which D- or L- form, especially its L- form), salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, thiocyanic acid and undecylenic acid, etc.

[0015] The salt of GL-V8 exists in the form of an organic solvate, a hydrate or a mixture of an organic solvate / hydrate; it is in the form of an organic solvate, for example, in the form of an ethanolate; it is in the form of a mixture of a hydrate / organic solvate, for example, in the form of a mixture of a hydrate / ethanolate.

[0016] The salt of GL-V8 exists in the form of crystals, partially crystals, amorphous or polymorphic crystals.

[0017] The structure of the salt of GL-V8 is mainly selected from the following:

[0018]

[0019] In a second aspect, the present invention provides a method for preparing a salt of GL-V8, comprising dissolving 5-hydroxy-7-(4-(bis(2-hydroxyethyl)amino)-1-yl)butoxy-8-methoxyflavone in a solvent, then dropping or adding a pharmaceutically acceptable acid once or multiple times, stirring to crystallize, and recovering the crystals to obtain the salt.

[0020] Furthermore, seed crystals are added during crystallization to carry out the reaction.

[0021] According to the present invention, preferably, the solvent is selected from: one or more of alcohols, ethers, esters, and halogenated alkanes.

[0022] Preferably according to the present invention, the alcohol is a C1-C10 alcohol, preferably a C1-C8 alcohol, and more preferably a C1-C5 alcohol.

[0023] Preferably according to the present invention, the alcohol is selected from: methanol, ethanol, n-propanol, isopropanol, n-butanol or a combination thereof.

[0024] Preferably according to the present invention, the ethers are C2-C8 ethers, preferably C2-C5 ethers.

[0025] Preferably according to the present invention, the ether is selected from: diethyl ether, tetrahydrofuran or a combination thereof.

[0026] Preferably according to the present invention, the esters are C1-C10 esters, preferably C1-C7 esters, and more preferably C1-C5 esters.

[0027] Preferably according to the present invention, the ester is selected from: methyl formate, ethyl acetate, isobutyl formate, isopropyl acetate or a combination thereof.

[0028] Preferably according to the present invention, the halogenated alkanes are C2-C8, preferably C2-C5 alkanes.

[0029] Preferably according to the present invention, the halogenated alkanes are selected from: dichloromethane, chloroform, carbon tetrachloride.

[0030] According to the preferred embodiment of the present invention, the molar ratio of 5-hydroxy-7-(4-(bis(2-hydroxyethyl)amino)-1-yl)butoxy-8-methoxyflavone to the pharmaceutically acceptable acid is 1:0.8-1:1.5, preferably 1:0.9-1:1.3, and more preferably 1:1.0-1:1.1.

[0031] Preferably according to the present invention, the temperature range of the crystallization process is 10-80°C, preferably 25-30°C; the reaction time is 0.1-10h, preferably 0.5-6h.

[0032] Preferably according to the present invention, the drying temperature in the recovery crystallization process is 10-90°C, preferably 20-80°C, and more preferably 40-70°C; the drying time is 5-150h, preferably 20-100h, and more preferably 24-60h.

[0033] Preferably according to the present invention, the purity of the product of the method is 50%-99.9%, more preferably 75%-99.9%, and even more preferably 85%-99.9%.

[0034] In a third aspect, the present invention provides a pharmaceutical composition comprising the salt of GL-V9 and a pharmaceutically acceptable carrier and / or diluent.

[0035] In a fourth aspect, the present invention provides the use of the salt of GL-V9 or a pharmaceutical composition thereof for preparing a drug for preventing or treating liver fibrosis, sepsis, colitis or cancer.

[0036] The cancers include gastric cancer, lung cancer, breast cancer, prostate cancer, leukemia, ovarian cancer, esophageal cancer, liver cancer, colon cancer, and brain cancer.

[0037] The GL-V8 salt type, especially the hydrochloride type, prepared by the present invention can exist stably, and has advantages in physical and chemical properties, preparation processing performance and bioavailability, such as melting point, solubility, hygroscopicity, stability, adhesion, compressibility, fluidity, in vitro and in vivo dissolution, in vitro efficacy, and biological effectiveness.

[0038] The present invention has the following beneficial effects: (1) the GL-V8 salt type (hydrochloride) prepared by the present invention has low hygroscopicity, and the low hygroscopicity can ensure that the sample can maintain low moisture weight gain without deliquescence during the later production, processing, storage and transportation, thereby ensuring the stability of the drug quality; at the same time, the solubility of the hydrochloride in water is higher than that of the base, which is conducive to the subsequent drug preparation.

[0039] (2) The GL-V8 salt type prepared by the present invention has high chemical stability under high temperature and high humidity conditions.

[0040] (3) The present invention provides a method for preparing a salt form of GL-V8. In the prior art, during the development of the production process of GL-V8, the free base has a strong electrostatic effect, which is not convenient for the expanded production of the preparation. The present invention uses a salt-forming method, and the electrostatic effect of the salt-formed GL-V8 is weak, which is conducive to large-scale industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 The growth inhibition curves of different GL-V8 salt types on human hepatoma cell MHCC-97H.

[0042] Figure 2 The growth inhibition curves of different GL-V8 salt types on human colon cancer cell HCT 116.

[0043] Figure 3 The growth inhibition curves of different GL-V8 salt types on human acute monocytic leukemia THP-1 cells.

[0044] Figure 4 The growth inhibition curves of different GL-V8 salt types on human pancreatic cancer cells BXPC3.

[0045] Figure 5 The growth inhibition curves of different GL-V8 salt types on human non-small cell lung cancer cells H460.

[0046] Figure 6 The growth inhibition curves of different GL-V8 salt types on human gastric cancer cell BGC-823.

[0047] Figure 7 The graph shows the growth inhibition curve of different GL-V8 salt types on human glioma U87 cells. DETAILED DESCRIPTION

[0048] The present invention is described in detail with reference to the following examples, which describe in detail the preparation and use of the salt forms of the present invention, but the scope of the present invention is not limited thereto. The experimental methods in the following examples are conventional methods unless otherwise specified. It is obvious to those skilled in the art that many changes to both materials and methods may be implemented without departing from the scope of the present invention.

[0049] Synthesis of Compounds of Formula I

[0050] The compound of formula I described in the present invention is 5-hydroxy-7-(4-(bis(2-hydroxyethyl)amino)-1-yl)butoxy-8-methoxyflavone.

[0051]

[0052] The free base 5-hydroxy-7-(4-(bis(2-hydroxyethyl)amino)-1-yl)butoxy-8-methoxyflavone was prepared by the preparation method described in patent application number CN115160279.

[0053] Example 1: Preparation of 5-hydroxy-7-(4-(bis(2-hydroxyethyl)amino)-1-yl)butoxy-8-methoxyflavone hydrochloride

[0054] Dissolve 2.0 g of 5-hydroxy-7-(4-(bis(2-hydroxyethyl)amino)-1-yl)butoxy-8-methoxyflavone in 20 ml of ethanol, cool to 5±5°C, slowly add 1.2 equivalents of hydrochloric acid (2 mol / L ethanol solution), the solution changes from turbid to clear, then stir at 20±5°C for 1 hour, a large amount of yellow solid precipitates, and filter to obtain hydrochloride. The obtained solid is placed in a vacuum drying oven and vacuum dried at 50°C for 24 hours to obtain 2.0 g of 5-hydroxy-7-(4-(bis(2-hydroxyethyl)amino)-1-yl)butoxy-8-methoxyflavone hydrochloride as a yellow solid powder.

[0055] Example 2: Preparation of the sulfate salt of 5-hydroxy-7-(4-(bis(2-hydroxyethyl)amino)-1-yl)butoxy-8-methoxyflavone

[0056] Dissolve 2.0 g of 5-hydroxy-7-(4-(bis(2-hydroxyethyl)amino)-1-yl)butoxy-8-methoxyflavone in 20 ml of ethanol, cool to 5±5°C, slowly add 1.2 equivalents of sulfuric acid (2 mol / L ethanol solution), the solution changes from turbid to clear, then stir at 20±5°C for 1 hour, a large amount of yellow solid precipitates, and the sulfate is filtered out. The obtained solid is placed in a vacuum drying oven and vacuum dried at 50°C for 24 hours to obtain 2.1 g of 5-hydroxy-7-(4-(bis(2-hydroxyethyl)amino)-1-yl)butoxy-8-methoxyflavone hydrochloride as a yellow solid powder.

[0057] Example 3: Preparation of 5-hydroxy-7-(4-(bis(2-hydroxyethyl)amino)-1-yl)butoxy-8-methoxyflavone hydrobromide

[0058] Dissolve 2.0 g of 5-hydroxy-7-(4-(bis(2-hydroxyethyl)amino)-1-yl)butoxy-8-methoxyflavone in 20 ml of ethanol, cool to 5±5°C, slowly add 1.2 equivalents of hydrobromic acid (48% aqueous solution), the solution changes from turbid to clear, then stir at 20±5°C for 1 hour, a large amount of yellow solid precipitates, and the hydrobromide is obtained by filtration. The obtained solid is placed in a vacuum drying oven and vacuum dried at 50°C for 24 hours to obtain 2.1 g of 5-hydroxy-7-(4-(bis(2-hydroxyethyl)amino)-1-yl)butoxy-8-methoxyflavone hydrobromide as a yellow solid powder.

[0059] Example 4: Preparation of 5-hydroxy-7-(4-(bis(2-hydroxyethyl)amino)-1-yl)butoxy-8-methoxyflavone acetate

[0060] Dissolve 2.0 g of 5-hydroxy-7-(4-(bis(2-hydroxyethyl)amino)-1-yl)butoxy-8-methoxyflavone in 20 ml of ethanol, cool to 5±5°C, slowly add 1.2 equivalents of acetic acid, the solution changes from turbid to clear, then stir at 20±5°C for 1 hour, a large amount of yellow solid precipitates, and the acetate is obtained by filtration. The obtained solid is placed in a vacuum drying oven and vacuum dried at 50°C for 24 hours to obtain 1.8 g of 5-hydroxy-7-(4-(bis(2-hydroxyethyl)amino)-1-yl)butoxy-8-methoxyflavone acetate as a yellow solid powder.

[0061] Example 5: Preparation of methanesulfonate of 5-hydroxy-7-(4-(bis(2-hydroxyethyl)amino)-1-yl)butoxy-8-methoxyflavone

[0062] Dissolve 2.0 g of 5-hydroxy-7-(4-(bis(2-hydroxyethyl)amino)-1-yl)butoxy-8-methoxyflavone in 20 ml of ethanol, cool to 5±5°C, slowly add 1.2 equivalents of methanesulfonic acid, the solution changes from turbid to clear, then stir at 20±5°C for 1 hour, a large amount of yellow solid precipitates, and methanesulfonate is obtained by filtration. The obtained solid is placed in a vacuum drying oven and vacuum dried at 50°C for 24 hours to obtain 1.5 g of methanesulfonate of 5-hydroxy-7-(4-(bis(2-hydroxyethyl)amino)-1-yl)butoxy-8-methoxyflavone as a yellow solid powder.

[0063] Example 6: Preparation of p-toluenesulfonate of 5-hydroxy-7-(4-(bis(2-hydroxyethyl)amino)-1-yl)butoxy-8-methoxyflavone

[0064] Dissolve 2.0 g of 5-hydroxy-7-(4-(bis(2-hydroxyethyl)amino)-1-yl)butoxy-8-methoxyflavone in 20 ml of ethanol, cool to 5±5°C, slowly add 1.2 equivalents of p-toluenesulfonic acid, the solution changes from turbid to clear, then stir at 20±5°C for 1 hour, a large amount of yellow solid precipitates, and p-toluenesulfonate is obtained by filtration. The obtained solid is placed in a vacuum drying oven and vacuum dried at 50°C for 24 hours to obtain 1.6 g of p-toluenesulfonate of 5-hydroxy-7-(4-(bis(2-hydroxyethyl)amino)-1-yl)butoxy-8-methoxyflavone as a yellow solid powder.

[0065] Experimental Example 1: Investigation of the hygroscopicity of GL-V8 salt

[0066] About 1000 mg of the GL-V8 salt prepared in Examples 1-6 were weighed respectively, placed at 25±5° C. and 80% relative humidity for 24 hours, and the weights of the samples before and after were compared. The details are shown in Table 1 below.

[0067] Regarding the description of hygroscopic characteristics and the definition of hygroscopic weight gain (Chinese Pharmacopoeia 2020 Edition General Chapter 9103 Drug Hygroscopicity Experiment Guidance Principles, Experimental Conditions: 25±1℃, 80% relative humidity, 24 hours):

[0068] Deliquescent: Absorbs enough water to form a liquid

[0069] Highly hygroscopic: weight gain due to moisture absorption is not less than 15.0%

[0070] Hygroscopic: Weight gain due to moisture absorption is less than 15.0% but not less than 2.0%

[0071] Slightly hygroscopic: weight gain due to moisture absorption is less than 2.0% but not less than 0.2%

[0072] No or almost no hygroscopicity: weight gain due to moisture is less than 0.2%

[0073] Table 1 Hygroscopicity of GL-V8 salt

[0074]

[0075] The results show that among the GL-V8 salt types, hydrochloride, sulfate, hydrobromide and acetate have lower hygroscopicity. The weight gain range of the hydrochloride type is: the weight gain due to moisture is less than 0.2%, which is no or almost no hygroscopicity; the weight gain range of the sulfate, hydrobromide and acetate types is: the weight gain due to moisture is less than 2.0% but not less than 0.2%, which is slightly hygroscopic. The weight gain range of the methane sulfonate and p-toluene sulfonate types is: the hygroscopicity is less than 15% but not less than 2%, which is hygroscopic, and the research on these two salt types will be abandoned in subsequent studies. The salt types (hydrochloride, sulfate, hydrobromide and acetate) of the present invention have low hygroscopicity, especially the hydrochloride, which indicates that the above salt types are not prone to deliquescence during drug production and storage.

[0076] Experimental Example 2: Salt Stability Study of GL-V8

[0077] A certain amount of the sample to be tested was weighed and placed in a watch glass, and placed at 25°C, 60% RH and 40°C, 75% RH, respectively. After 30 days, the purity was determined by HPLC. The experimental results are shown in Table 2 below, and the HPLC purity test method is shown in Table 3.

[0078] Table 2 Salt stability of GL-V8

[0079]

[0080] Table 3 HPLC related substances detection method

[0081]

[0082]

[0083] Solution preparation: Take 1 mg of sample and dissolve it in 1 ml of methanol by ultrasonic.

[0084] The results show that the salt of GL-V8 of the present invention has high chemical stability and can maintain physical and chemical stability for at least 1 month.

[0085] Experimental Example 3: Study on the solubility of GL-V8 salt in water

[0086] Use a pipette to take 1 mL of pure water and add it to a 1.5 mL glass vial. Weigh an appropriate amount of the salts prepared in Examples 1-4 and place them in a glass vial to prepare a supersaturated aqueous solution. Ultrasonicate for 30 min at room temperature. Take the supernatant and determine the content using a standard curve. Test the dissolution. The water solubility results of each salt are shown in Table 4 below:

[0087] Table 4 Test results of solubility of GL-V8 salt in water

[0088] Salt type Water solubility (mg / ml) GL-V8 Base 10.05 Hydrochloride 16.78 Sulfate 8.66 Hydrobromide 12.23 Acetate 20.45

[0089] The results showed that the hydrochloride and acetate salts had higher water solubility, which was significantly improved compared with the free state.

[0090] Experimental Example 4: Growth inhibitory activity test on human tumor cells

[0091] Experimental Materials

[0092] 1. Cells

[0093] Human hepatoma cell line MHCC-97H, human colon cancer cell line HCT 116, human acute monocytic leukemia cell line THP-1, human non-small cell lung cancer cell line H460, human pancreatic cancer cell line BXPC3, human gastric cancer cell line BGC-823 and human glioma cell line U87 were purchased from the Institute of Cell Biology, Chinese Academy of Sciences, Shanghai, and cultured in MEM, DMEM and RPMI 1640 medium containing 10% fetal bovine serum at 37°C in a humidified cell culture incubator containing 5% CO2.

[0094] 2. Reagents

[0095] (1) Cell culture medium: GIBCO, USA. Store in a refrigerator at 4°C away from light.

[0096] (2) Fetal bovine serum: Vicente Biotech, Canada. Inactivate in a 56°C water bath for 30 min before use to remove endotoxins, etc., and store at 4°C after aliquoting.

[0097] (3) Dimethyl sulfoxide (DMSO): Sigma-Aldrich, USA. Store at room temperature away from light.

[0098] (4) Methylthiazolyl tetrazolium (MTT) solution: Fluka, USA. Weigh 250 mg of MTT and add it to 50 mL of 0.01 M PBS (pH 7.2) buffer. Ultrasonicate at room temperature for 1 h until the solution becomes clear and transparent to prepare a 5 mg / mL MTT solution. Filter through a 0.22 μm microporous filter to sterilize the solution and store at -80°C in the dark.

[0099] Experimental instruments

[0100] 1. Model 3111 water-jacketed CO2 incubator: Thermo Corporation, USA.

[0101] 2. YJ-875 medical cleanroom workbench: Suzhou Purification Equipment Factory.

[0102] 3. Varioskan full wavelength microplate reader: Thermo Fisher Scientific, USA.

[0103] Experimental methods

[0104] In vitro cell culture

[0105] Human tumor cells were inoculated into cell culture flasks, and cell culture medium containing 10% fetal bovine serum was used. The cells were cultured at 37°C in a humid cell culture incubator containing 5% CO2. After the cells grew to an adherent state, the culture medium was discarded, and the cells were rinsed twice with 0.25% EDTA solution, 2-3 mL each time. After discarding the excess EDTA, 1 mL of 0.05% trypsin-EDTA solution was added, rinsed and poured out, and the cells were placed in a cell culture incubator for digestion. During this process, a new culture flask could be prepared, 4.5 mL of fresh culture medium was added, and digestion was terminated with 2 mL of fresh culture medium until most of the cells fell off. The cells were then inoculated into a new cell culture flask according to the cell density required for the experiment.

[0106] 2. Cell viability detection (MTT colorimetric assay)

[0107] The MTT method was used to evaluate the inhibitory effect of the test compound on the in vitro growth of human tumor cell lines. The mitochondria of living cells can reduce MTT to water-insoluble purple crystalline formazan that is deposited in the cells, and DMSO can dissolve the crystals. Therefore, after dissolving with DMSO, the absorbance value can be detected at a wavelength of 570nm using an enzyme marker. The number of living cells is proportional to the absorbance value within a certain range.

[0108] Seven human tumor cells in the logarithmic growth phase (human liver cancer cell line MHCC-97H, human colon cancer cell line HCT116, human acute myeloid leukemia cell line THP-1, human lung cancer cell line H460, human pancreatic cancer cell line BXPC3, human gastric cancer cell line BGC-823 and human glioma cell line U87) were inoculated in a 96-well plate at a density of 2500-4000 cells / well, with 5 duplicate wells in each group. Drug administration began 24 hours after inoculation. The concentrations of the test compounds were intervened in the cells at gradient concentrations (intervention concentration range was 0.75μM-30μM) for 24 hours, and then 20μL of MTT (5mg / mL) solution was added to each well. The cells were incubated in a light-proof cell culture incubator at 37°C for 4 hours, the supernatant was discarded with a 1mL syringe, 100μL of DMSO was added to each well to dissolve the bottom crystals, and the absorbance value was detected at 570nm.

[0109] Cell growth inhibition rate (Inhibition rate) % = (1-(xμM absorbance-blank group absorbance) / (0μM absorbance-blank group absorbance)) × 100%

[0110] 3. Data statistics and analysis

[0111] The experimental data were expressed as Mean ± SD. The statistical software was GraphPad Prism 9.5.

[0112] Experimental Results

[0113] 1. IC50 (μM) for 7 human tumor cell lines

[0114] Table 5 IC50 (μM) of GL-V8 salts against human tumor cell lines

[0115]

[0116] 2. Growth inhibition curves of 7 human tumor cell lines are shown in Figure 1-7 shown.

[0117] The results showed that the above-mentioned GL-V8 salts exhibited strong inhibitory activity against 7 human tumor cell models, among which GL-V8 hydrochloride exhibited significantly better inhibitory activity than GL-V8 base in human pancreatic cancer cells BXPC3 and human brain glioma cells U87.

[0118] The above shows and describes the basic principles, main features and advantages of the present invention. However, the above is only a specific embodiment of the present invention, and the technical features of the present invention are not limited thereto. Any other implementation methods derived by any technician in the field without departing from the technical solution of the present invention should be included in the patent scope of the present invention.

Claims

1. A salt of GL-V8, characterized in that The salt of GL-V8 is a salt formed by 5-hydroxy-7-(4-(bis(2-hydroxyethyl)amino)-1-yl)butoxy-8-methoxyflavone and a pharmaceutically acceptable acid in a stoichiometric ratio of 1:

1.

2. The salt of GL-V8 according to claim 1, characterized in that The pharmaceutically acceptable acid is selected from hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid, sulfuric acid, acetic acid, 2,2-dichloroacetic acid, adipic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, benzoic acid, 4-acetamido-benzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecyl sulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxy-ethanesulfonic acid, formic acid, galactaric acid, gentisic acid , glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, thiocyanic acid and undecylenic acid.

3. The salt of GL-V8 according to claim 1, characterized in that The salt of GL-V8 exists in the form of an organic solvate, a hydrate or a mixture of an organic solvate / hydrate.

4. The salt of GL-V8 according to claim 1, characterized in that The salt of GL-V8 exists in the form of crystal, partially crystal, amorphous or polymorphic crystal.

5. The salt of GL-V8 according to claim 1, characterized in that The structural formula of the salt of GL-V8 is selected from the following:

6. The method for preparing the salt of GL-V8 according to any one of claims 1 to 5, characterized in that: The method comprises the following steps: 5-Hydroxy-7-(4-(bis(2-hydroxyethyl)amino)-1-yl)butoxy-8-methoxyflavone is dissolved in a solvent, and then a pharmaceutically acceptable acid is added dropwise or added once or multiple times, stirred for crystallization, and the crystals are recovered to obtain the product.

7. The method for preparing the salt of GL-V8 according to claim 6, characterized in that: During crystallization, seed crystals are added to allow the reaction to proceed.

8. The method for preparing the salt of GL-V8 according to claim 6, characterized in that: The solvent is selected from one or more of alcohols, ethers, esters, and halogenated alkanes.

9. A pharmaceutical composition, characterized in that Comprising the salt of GL-V8 according to any one of claims 1 to 5 and a pharmaceutically acceptable carrier and / or diluent.

10. Use of the salt of GL-V8 according to any one of claims 1 to 5 or the pharmaceutical composition according to claim 9 in the preparation of a drug for preventing or treating liver fibrosis, sepsis, colitis or cancer.

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